Inhibition of mitochondrial complex I reverses NOTCH1-driven metabolic reprogramming in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is commonly driven by activating mutations in NOTCH1 that facilitate glutamine oxidation. Here we identify oxidative phosphorylation (OxPhos) as a critical pathway for leukemia cell survival and demonstrate a direct relationship between NOTCH1, elevated OxPhos gene expression, and acquired chemoresistance in pre-leukemic and leukemic models. Disrupting OxPhos with IACS-010759, an inhibitor of mitochondrial complex I, causes potent growth inhibition through induction of metabolic shut-down and redox imbalance in NOTCH1-mutated and less so in NOTCH1-wt T-ALL cells. Mechanistically, inhibition of OxPhos induces a metabolic reprogramming into glutaminolysis. We show that pharmacological blockade of OxPhos combined with inducible knock-down of glutaminase, the key glutamine enzyme, confers synthetic lethality in mice harboring NOTCH1-mutated T-ALL. We leverage on this synthetic lethal interaction to demonstrate that IACS-010759 in combination with chemotherapy containing L-asparaginase, an enzyme that uncovers the glutamine dependency of leukemic cells, causes reduced glutaminolysis and profound tumor reduction in pre-clinical models of human T-ALL. In summary, this metabolic dependency of T-ALL on OxPhos provides a rational therapeutic target.

The box plot presents the 25th to 75th percentiles with the median value highlighted and whiskers marking the minimum and maximum value. (Log2 transformed data; Wilcoxon rank sum test: P = 10 −8 , left panel; P = 8.6 × 10 −6 , right panel). b) Genes that were upregulated by the NOTCH1 oncogene (ICN) in CD4 + CD8 + DP cells were analyzed for pathway enrichment (FDR ≤ 0.1, GSE12948). Shown are the FDR q values (−log10) for overlaps computed with the reference datasets in MSigDB. c) Gene set enrichment analysis (GSEA) of gene set from AALL1231 trials (N=75), indicated enrichment of genes related to glutamine metabolism and mitochondrial metabolism as well as translation and downregulation of apoptosis-related genes in patients with NOTCH1 mutations (each vertical bar in x axis is gene rank in the pathway list and y axis represents running enrichment score). d) The OxPhos gene signature was significantly enriched in T-ALL patients with NOTCH1 mutations in the TARGET cohort of 265 patients (b) and the COG AALL1231 cohort of 75 patients (f); two-sided t-test; The center line represents the median and whiskers represents maximum (Q3 + 1.5*IQR) and minimum value (Q1 + 1.5*IQR); e) Patient samples with NOTCH1 mutations were characterized by significant enrichment of mitochondrial translation genes in both the TARGET (c) and COG AALL1231 (g) cohorts; two-sided t-test; The center line represents the median and whiskers represents maximum (Q3 + 1.5*IQR) and minimum value (Q1 + 1.5*IQR); f) Gene expression analysis in the TARGET (d) and COG AALL1231 (h) cohorts showed lower enrichment of apoptosis-related genes in patients with NOTCH1 mutations than in those with wild-type NOTCH1. two-sided t-test; The center line represents the median and whiskers represents maximum (Q3 + 1.5*IQR) and minimum value (Q1 + 1.5*IQR); g) Venn diagrams enriched by over representation analysis of differentially expressed genes, and Volcano plots showing cancer dependencies associated with OxPhos-related genes, graphed as P value (-log10, y-axis) against effect size (x-axis), comparing T-ALL versus other hematological malignancies ( left) and T-ALL versus other cancers (right), two-sided t-test; Supplementary Figure 2. NOTCH1 activation affects the response to OxPhosinhibition of primary pre-LSCs. a) Notch1 gene status in SCL tg LMO1 tg preleukemic and leukemic thymocytes. Notch1 gene activating mutations and their impact on protein sequences are shown. PEST, protein degradation domain; b) Dose-response analysis of pre-LSCs to IACS-010759. Dose-response curves shown in Figures 2C were analyzed by non-linear curve fitting, to compute the maximum inhibition and the EC50 for the indicated genotypes under MS5-DL4 or MS5 co-culture conditions (mean ± SD, n=3 independent experiments).two-way ANOVA; p-value ****<0.0001, c) Cell death analysis of LMO1 tg pre-LSCs co-cultured on MS5-DL4 stromal cells following drug treatment (% of early apoptotic, late apoptotic and necrotic cells at 24-72 h) (mean ± SD, n=3 independent experiments). Two-way ANOVA, p-values *=0.045; **=0.0013; ***=0.0002; and ****<0.0001, d) Cell death analysis of SCL tg LMO1 tg pre-LSCs co-cultured on MS5-DL4 stromal cells following drug treatment (% of early apoptotic, late apoptotic and necrotic cells at 24-72 h) (mean ± SD, n=3 independent experiments). e) Viability analysis of MS5-DL4 stromal cells following drug treatment. MS5-DL4 stromal cells were grown in presence of different concentrations of IACS-010759. Shown is the percentage of living MS5-DL4 stromal cells. f) Representative DIC image of cell cultures after 48h treatment with the indicated dose of IACS-010759, scale bar 20 uM, from n=3 independent experiments .

Supplementary Figure 4. IACS-010759 inhibits Oxygen consumption rate in NOTCH1 wild type and mutant T-ALL cell lines a)
Representative graphs of Oxygen consumption rate (OCR) (left) and extracellular acidification rate (ECAR) (right) response during Mito Stress Test in NOTCH1-wt T-ALL cell lines after 4 h treatment with the indicated concentrations of IACS-010759 (mean±SD, n=3 independent experiments, n=4 replicates/condition).

b)
Representative OCR (left) and ECAR (right) during Mito Stress Test in NOTCH1mutated T-ALL cell lines after 4 hr treatment with the indicated concentrations of IACS-010759 (mean±SD, n=3 independent experiments, n=4 replicates/condition).

Supplementary Figure 5. DLL4 does not impact response to OxPhos-i in Tlymphocytes a)
Correlation of viability reduction and decrease in OCR in T-ALL cell lines and Tlymphocytes exposed to IACS-010759 at 10 nM; simple liner regression: F=10.11; DFn,DFd=1,12; P value=0.0079; R squared =0.4572 b) Correlation analysis of cell viability reduction after treatment with IACS-010759 and increase in ROS across T-ALL cell lines and T-lymphocytes; (mean±SD, n=3 independent experiment); simple linear regression F=15.21; DFn,DFd=1,12; P-value=0.0021; R squared =0.5590; c) Oxygen consumption rate (OCR) measured on healthy T-lymphocytes following culture with or without DLL4, treated with DMSO or 10 nM of IACS-010759; Mito Stress Test assay, by Seahorse;(mean±SD, n=4 replicates/condition) d) Extracellular acidification rate (ECAR) measured on healthy T-lymphocytes following culture with or without DLL4, treated with DMSO or 10 nM of IACS-010759; Mito Stress Test assay, by Seahorse; (mean±SD, n=4 replicates/condition) e) Basal OCR and basal ECAR from the experiment described in (C) and (D) (mean±SD, n=3 independent donors, n=4 replicates/condition), ns-no significance; one-way ANOVA; f) Maximal OCR and maximal ECAR from the experiment described in (C) and (D) (mean±SD, n=3 independent donors, n=4 replicates/condition), ns-no significance; oneway ANOVA; g) Representative Western blots of protein expression changes over 24 h after treatment with 10 nM IACS-010759 in the NOTCH1-mutated cell line JURKAT. Primary antibodies against proteins involved in the mTOR/Akt pathway, AMPK pathway, glycolysis pathway, DNA damage response, and autophagy were used, results representative of n=3 experimental replicates. h) Top: KEGG signaling pathways affected by IACS-019759 in NOTCH1-mutated cell line PF-382; Bottom: Gene Ontology analysis of genes affected by IACS-010759 treatment.

Supplementary Figure 7. NOTCH1 controls glycolytic capacity upon OxPhos blockade a)
Representative graphs of Oxygen consumption rate (OCR) (left) and extracellular acidification rate (ECAR) (right) during Glycolysis Stress Test in NOTCH1-wt T-ALL cell lines after 4 hr treatment with the indicated concentrations of IACS-010759 (mean±SD, n=3 independent experiments, n=4 replicates/condition). b) Representative graphs of OCR (left) and ECAR (right) during Glycolysis Stress Test in NOTCH1-mutated T-ALL cell lines after 4 hr treatment with the indicated concentrations of IACS-010759 (mean±SD, n=3 independent experiments with n=4 replicates/condition).

Supplementary Figure 8. NOTCH1 status impacts ATP generation upon OxPhos blockade a)
Representative graphs of OCR (left) and ECAR (right) during Real Time ATP Assay in NOTCH1-wt T-ALL cell lines after 4 hr treatment with the indicated concentrations of IACS-010759 (mean±SD, n=3 independent experiments, n=4 replicates/condition). b) OCR (left) and ECAR (right) during Real Time ATP Assay in NOTCH1-mutated T-ALL cell lines after 4 hr treatment with the indicated concentrations of IACS-010759 (mean±SD, n=3 independent experiments, with n=4 replicates/condition).

Supplementary Figure 9. IACS-010759 inhibits TCA cycle in time-and dosedependent manner a)
Modulation of selected intracellular metabolites in NOTCH1-mutated T-ALL cell line PF-382 and NOTCH1-wild type cell line SUP-T1 after treatment with IACS-010759 or DMSO for 24 hrs. Levels shown are expressed as log2 of the ratio of metabolite levels in cells treated with IACS-010759 to metabolite levels in cells treated with DMSO; b) Intracellular modulation of selected metabolites following treatment with IACS-010759 in the NOTCH1-mutated cell lines JURKAT and PF-382. Cells were treated for 12 and 24 hrs with 0 nM, 13 nM, or 123 nM IACS-010759. Intracellular metabolite extracts were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry. Concentrations were normalized to the average metabolite level in the control samples (treated with 0 nM IACS-010759) at 12 h. Mean ± SD (4 replicates) relative concentrations are shown. Significance was determined using multiple comparisons two-way ANOVA; ns-no significant, p-values are shown * < 0.05; ** < 0.01; *** = 0.0001; **** < 0.0001; c) Mass spectrometry measured α-ketoglutarate and citrate levels and the ratio of αketoglutarate/citrate, at 2, 4, 6 and 12 hr and upon treatment with DMSO, 13 or 123 nM of IACS-010759 (mean±SD, n=1 with 4 technical replicates, for two cell lines JURKAT and PF382). Significance was determined using a one-sided Student t-test (to compare either 13 or 123 nM IACS-010759 versus control) followed by the false discovery rate (FDR). The changes over time were compared using two-way ANOVA, p-values are shown as: * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001;

Supplementary Figure 10. Glucose labelled flux analysis shows changes in TCA cycle activity upon OxPhos blockade in NOTCH1 mutant cell line PF-382
Effects of IACS-010759 at a dose of 10 nM after 12 hrs on the metabolic fluxes in NOTCH1-mutated T-ALL cell line PF-382 cultured with 13 C6 glucose were analyzed using stable isotope-resolved metabolomics and ultra-performance liquid chromatographytandem mass spectrometry.

Supplementary Figure 11. Glucose and Glutamine labelled flux analysis shows changes in TCA cycle activity and utilization of reductive metabolism of glutamine upon OxPhos blockade in NOTCH1 wild type cell line SUP-T1
Effects of IACS-010759 at a dose of 10 nM on the metabolic fluxes in NOTCH1-wild type T-ALL cell line SUP-T1 cultured with (A) 13 C6 glucose or (B) 13 C5, 15 N2-glutamine were analyzed using stable isotope-resolved metabolomics and ultra-performance liquid chromatography-tandem mass spectrometry.

Supplementary Figure 17. Combination of IACS-010759 and CB-839 shows enhanced blockade of TCA cycle in NOTCH1 mutant T-ALL
Stable isotope-resolved metabolomics and ultra-performance liquid chromatographytandem mass spectrometry analysis of NOTCH1-mutated T-ALL cell line PF-382 labelled with 13 C6-glucose and exposed to treatment with the combination of 10 nM IACS-010759 and 1 μM CB-839 for 12 h.

Supplementary Figure 18. Combination of IACS-010759 and CB-839 shows enhanced blockade of TCA cycle in NOTCH1 wild type T-ALL a) Stable isotope-resolved metabolomics (SIRM) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis of NOTCH1-wild
type T-ALL cell line SUP-T1 labelled with 13 C6-glucose and exposed to treatment with the combination of 10 nM IACS-010759 and 1 μM CB-839 for 24 h. b) SIRM and UPLC-MS/MS analysis of NOTCH1-wild type T-ALL cell line SUP-T1 labelled with 13 C5, 15 N2-glutamine and exposed to treatment with the combination of 10 nM IACS-010759 and 1 μM CB-839 for 24 h.

Supplementary Figure 20. Gen expression analysis indicates deep metabolic changes upon dual OxPhos-and GLS-blockade a)
Top: KEGG signaling pathways affected by IACS-010759 in combination with CB-839 in the NOTCH1-mutated cell line PF-382. Bottom: Gene Ontology analysis of genes affected by combination of IACS-010759 and CB-839. b) Heatmap for gene set enrichment analysis (GSEA) displays differences in enrichment of genes related to glutamine metabolism, mitochondria translation and transcription, respiration and apoptosis analyzed by KEGG and displayed as a z-score affected by IACS-010759, CB-839 or combination of both compounds in the NOTCH1-mutated cell line PF-382.

Supplementary Figure 22. Flow cytometry analysis indicate presence of circulating tumor cells prior initiation of treatment in mice harboring Notch1 mutated murine T-ALL leukemia, that undergo profound metabolic changes following treatment initiation. a)
Flow cytometry analysis of murine blood on day 7 post transplantation. After gating out debris and dead cells (DAPI+), double positive: GFP+, mCD45+ leukemic cells were identified on a GFP (FITC) (y-axis)-vs mCD45 (APC) (x-axis)-gated contour plot. b) The average level of engraftment in BL6 mice harboring GLS fl/fl leukemia prior to treatment initiation: % of gated double positive cells were analyzed and divided by % of all mCD45+ cells and multiplied by 100% to obtain normalized leukemic engraftment, followed by mice randomization into 4 groups: GLS+, GLS+IACS-010759, GLS-, GLS-IACS010759 respectively (mean±SD, n=5 independent mice/ treatment group). c) Heatmap of mass spectrometry analysis of metabolites found in PB of mice transplanted with murine NOTCH1-mutated T-ALL cells after 5 days of treatment with vehicle; IACS-010759; tamoxifen to induce GLS knockout, or combination of both tamoxifen and IACS-010759, (n=3 for each treatment). Results are expressed as a mean log of fold-change ratio over the level of metabolites measured in mice treated with vehicle; d) Panel of common metabolites regulated in same way in vivo and in vitro.

Supplementary Figure 23. IACS-010759 in combination with VXL produces viability reduction at low nanomolar concentrations overcoming intrinsic Dexamethasone resistance a)
Representative graphs of OCR measured by Mito Stress Test for the NOTCH1mutated T-ALL cell line KOPT-K1 treated with (from left to right): dexamethasone, and/or IACS-010759; vincristine and/or IACS-010759, and L-asparaginase and/or IACS-010759 (mean±SD, n=3 independent experiments with n=4 replicates per condition). b) Analysis of cell viability under treatment with increasing doses of IACS-010759 (0-50 nM) and dexamethasone (0-370 nM), in the indicated T-ALL cell lines; c) Analysis of cell viability under treatment with increasing doses of IACS-010759 (0-50 nM) and vincristine (0-50 ng/mL), in the indicated T-ALL cell lines; d) Analysis of cell viability under treatment with increasing doses of IACS-010759 (0-50 nM) and L-asparaginase (0-5 IU), in the indicated T-ALL cell lines; e) Analysis of cell viability under treatment with increasing doses of IACS-010759 (0-50 nM) and VXL (0-15 ng/mL vincristine, 0-150 nM dexamethasone, 0-1.5 IU Lasparaginase) in the indicated T-ALL cell lines. Each square of the concentration matrix represents a mean value, normalized to that of DMSO-treated controls, from 5 independent experiments, as measured by CTG assay.

Supplementary Figure 24. VXL and IACS-010759 combination produces deeper reduction of OCR following VXL-driven upregulation of OCR in both NOTCH1 wild type and mutant T-ALL cell lines a)
Representative graphs of OCR and ECAR response determined by Mito Stress Test in NOTCH1-wild type T-ALL cell lines after treatment with DMSO, VXL, IACS-010759 and combination of VXL and IACS-010759. (mean±SD, n=3 independent experiments with n=4 replicates per condition) b) Representative graphs of OCR and ECAR response upon treatment with DMSO, VXL, IACS-010759 and combination of IACS-010759 and VXL determined by Mito Stress Test in NOTCH1-mutated T-ALL cell lines in response to treatment with VXL and/or IACS-010759 (mean±SD, n=3 independent experiments with n=4 replicates per condition).

Supplementary Figure 25. IACS-010759 and VXL reduce moderatelyoxygen consumption rate in healthy bone marrow cells but don't reduce colony forming unit capacity a)
Basal OCR represented as summaries of 6 cell lines with NOTCH1 mutation and 3 cell lines with NOTCH1 wild type subjected to treatment with IACS-010759 at 10 nM, VXL, or combination of both compounds versus control (DMSO) (mean±SD, n=3 independent experiment, with 4 replicates per each treatment condition); Two-way ANOVA; p -value: ns-no significant, *=0.0208; **=0.0019; ***=0.0002; ****<0.0001; b) Maximal OCR represented as summaries of 6 cell lines with NOTCH1 mutation and 3 cell lines with NOTCH1 wild type subjected to treatment with IACS-010759 at 10 nM, VXL, or combination of both compounds versus control (DMSO) (mean±SD, n=3 independent experiment, with 4 replicates per each treatment condition). Two-way ANOVA; p -value: ns-no significant, *=0.0106; **=0.0049; c) Oxygen consumption rate (OCR) (left) and extracellular acidification rate (ECAR) (right) determined by Mito Stress Test in healthy donor derived bone marrow cells treated with 24 h treatment with vehicle (control), 10 nM of IACS-010759, VXL or its combination; (n=2 independent hBM donors, (mean±SD, n=4 replicates per each condition); d) Analysis of ATP content of human bone marrow cells derived from healthy donor following treatment with VXL; 10 nM IACS01759 or combination of both for 96 hr; normalised to DMSO-treated controls, from 3 independent bone marrow donors, as measured by CTG assay; (mean±SD, n=3 replicates/condition); One-way ANOVA; pvalue: ns-no significant, *=0.0409; ****<0.0001; e) Comparison of results for the total number of colonies from colony forming unit assay (left) and presentation of colony units according to the lineage and comparison across the treatments (right); Healthy bone marrow cells were obtained fresh from healthy bone marrow donors and seeded at the density of 1 milion/ml for 24 hr with DMSO (CONTROL), 10 nM IACS01759, VXL or combination of both, following by collecting the cells and counting and seeding in Methylcellulose at the density of 25 k/ml for 12-14 days; colonies were counted manually from 3 independent dishes per consistency. (mean±SD); Oneway ANOVA; p -value: *=0.016; **=0.0044;

Supplementary Figure 27. SIRM and UPLC-MS/MS analysis indicate on-target blockade of both TCA cycle and glutaminolysis for T-ALL NOTCH1 wild type cell line SUP-T1 upon combination of VXL and IACS-010759 a)
Stable isotope-resolved metabolomics and ultra-performance liquid chromatographytandem mass spectrometry analysis of the NOTCH1-wild type T-ALL cell line SUP-T1 labelled with (A) 13C6-glucose or b) 13C5, 15N2-glutamine and treated with the combination of 10 nM IACS-010759 and VXL for 24 hrs. (mean±SD, n=3 replicates/condition), two-tailed Student t-test. c) Comparison of selected metabolites: glutamine, glutamate, Asparagine and aspartate measured in culture media collected from NOTCH1-wild type cell line SUPT1 cell culture after treatment with vehicle, IACS-010759, VXL or IACS-010759/VXL combination (mean±SD, n=1 independent experiment, with 3 replicates per each treatment condition). One-way ANOVA; p -value: *=0.0263; ****<0.0001;

Supplementary Figure 29. Impact on cell growth inhibition and apoptosis induction of combined IACS-010759/VXL blockade in vitro cant be reversed by NAC, but leads to profound metabolic changes as shown in circulating leukemic cells in vivo a)
Viability analysis of NOTCH1-mutated T-ALL cell lines JURKAT and PF382, following treatment with 10 nM IACS01759, VXL or combination of both for 72 hrs; in presence of N-Acetylcysteine (NAC) at the concentration of 0.5 or 2.0 mM respectively, normalised to DMSO-treated controls (by flow cytometry);(mean±SD, n=3 independent experiments), two-way ANOVA; P-values: **=0.0083; ***=0.00031; and ****<0.0001, b) Apoptosis evaluation by Annexin V-assay from the experiment described in (A); (mean±SD, n=3 independent experiments), two-way ANOVA; P-values: *=0.03; **=0.0096; ***=0.0008; ****<0.0001, c) ROS evaluation as expressed by H2DCFDA mean fluorescence intensity (MFI) measured by flow cytometry normalised to CONTROL (cells treated with DMSO) as described in (A). (mean±SD, n=3 independent experiment, with 3 replicates per each treatment condition), two-way ANOVA; P-values: *=0.02; **=0.0099; ****<0.0001, d) Heatmap of mass spectrometry analysis of metabolites found in PB of mice transplanted with murine Notch1-mutated PDX 80 T-ALL cells after 12 hr of treatment with vehicle; IACS-010759; VXL and combination of VXL and IACS-010759, (n=2 for each treatment). Results are expressed as a mean log of fold-change ratio over the level of metabolites measured in mice treated with vehicle; e) Ingenuity Pathway Analysis of metabolites presented as heatmap showing the most important metabolism-related canonical pathways altered under VXL and chemical inhibition of complex I, analyzed by Ingenuity Pathway Analysis software (QIAGEN);

Supplementary Figure 30. Mass spectrometry analysis of bone marrow cells derived from mice harboring PDX 80 indicate profound changes upon IACS-010759 and VXL dual intervention
Heatmap of mass spectrometry analysis of metabolites in bone marrow of mice transplanted with NOTCH1-mutated PDX80 after 12 hrs of treatment with vehicle, IACS-010759, VXL, or the combination of IACS-010759 and VXL (n=2 for each treatment);

Supplementary Figure 31. Mass spectrometry analysis of spleen cells derived from mice harboring PDX 80 indicate profound changes upon IACS-010759 and VXL dual intervention
Heatmap of mass spectrometry analysis of metabolites in spleens of mice transplanted with NOTCH1-mutated PDX80 after 12 hrs of treatment with vehicle, IACS-010759, VXL, or the combination of IACS-010759 and VXL (n=2 for each treatment);

Supplementary Figure 32. Ex vivo OCR measurement confirms reduction of oxidative respiration upon VXL and IACS-010759 treatment, blocking biosynthesis and bioenergetic processes in mitochondria in T-ALL PDX model D115 a)
Oxygen consumption rate (OCR) in spleen cells harvested from mice bearing D115, CU76, and PDX80 PDXs treated with vehicle (black), VXL (blue), IACS-010759 (red), or the combination of VXL with IACS-010759 (green) (mean±SD, each dot represents mean of 4 technical replicates for individual mice); P-values: **=0.0035; ***=0.0002; ****<0.0001, one-way ANOVA. b) Heatmap for gene set enrichment analysis (GSEA) displays differences in enrichment of genes related to glutamine metabolism, mitochondria translation and transcription, respiration and apoptosis analyzed by KEGG and displayed as a z-score affected by IACS-010759, VXL or combination of both compounds in the NOTCH1-mutated cell line PF-382.